Upon the determination of the concentration of metabolism parameters from body fluids in the field of clinical chemistry, an exact volumetric dosaging of sample and of dissolved reagents plays a decisive role. Generally there are employed for this dosaging aids of the type of pipettes which may depending on their nature, be glass or plunger pipettes, or fully or partially automated dosaging devices. These aids have the disadvantage that they are of high cost; they furthermore require continuous supervision in the sense of calibration verification in order to assure the correctness of the volume of liquid dosed. In the case of partially automatic and manually actuated systems the possibility of errors in operation can furthermore not be excluded so that operation by non-skilled personnel is out of the question.
It has therefore been successfully attempted to avoid the above-described difficulties by preportioning the individual components of the reagent mixture depending on the resultant stability, either in pure form or as partial mixture, and thus doing away with the necessity of pipetting steps for the user. For the dosaging of the specimen (blood, serum or plasma) there can furthermore be used a so-called end-to-end capillary which automatically fills itself on basis of capillary forces, whereby the danger of errors in dosaging can be excluded, or at least reduced.
If a reagent component or a mixture of several components is stable in solution, then the preportioning can be effected by provision of a volume of liquid supplied by machine. For this there is suitable, for instance, the buffer solution, which can advantageously be introduced directly into the measurement cell. Such a method, which in addition employs a specimen capillary is described in DE-OS 24 22 260 (West German Unexamined Application for Patent).
A pre-portioning, however, affords difficulties with respect to preparation if individual components are used in only very small quantities, such as enzymes, co-enzymes or chromogens. One way out of this difficulty is to mix the small amounts of active substance with a larger amount of inert filler and to work the mixture into a tablet, which can then be handled more easily.
However, this method of preparation is not without problems with respect to the dissolving time of the tablet and possible losses of active substance--for instance loss of enzymatic activity--upon the compressing of the mixture to form the tablet. It furthermore presupposes that the active substance be available, stable, in solid form, which is not always true, particularly in the case of enzymes--for example cholesteroloxidase.
In order to solve these difficulties it has been proposed to include such small amounts of substance in a solvent, fill a capillary with the resultant solution, and precipitate the substance by evaporating the solvent in the capillary. Such a coated capillary represents a particularly advantageous form of administration of the reagent component since it makes it possible easily to handle even very small amounts of substance without addition of fillers, which might prove disturbing. One method of this type is described in DE-OS 27 21 942 (West German Unexamined Application for Patent).
The coated capillaries must satisfy a number of requirements the satisfying of which is of importance for the possibility of use or the range of use. Thus the substance must be deposited in stable form without the active substance spoiling. The process must furthermore assure a readily reproducible coating with respect to the amount of substance and be suited for industrial mass production. Finally, it is advantageous for the capillary to be still absorptive even after the coating, so that it can be used for the dosing of further reagent components, for instance a liquid enzyme suspension.
The satisfying of these requirements encounters difficulties, particularly furthermore in the case of an important chromogen of clinical chemistry, namely 4-aminophenazone. Thus this substance tends to deposit from a large number of solvents upon evaporation of the solvent not in crystalline form but as an amorphous oil. The oil obtained begins to solidify after a short period of time forming a yellow color, and a resinous hydrophobic layer is produced by which the capillary loses its absorption capacity. The yellow coloration of the substance expresses itself in a new band in the absorption spectrum and has a disturbing effect on photometric measurement due to the occurrence of high reagent blank values. If the conditions under which the solvent is evaporated are varied, for instance by applying a vacuum, it is, it is true, possible in individual cases to obtain a finely crystalline precipitation with good absorptivity but it is found that these results are of uncertain reproducibility, so that mass production is not thereby possible.
Another difficulty is that even if a crystalline precipitation having in principle good adsorption properties is obtained, the substance may not deposit in a homogeneous layer but rather as a plug at one end of the capillary, whereby the capillary is closed up and therefore is no longer adsorptive.
The known method of freeze-drying is not suitable for overcoming the above-indicated difficulties since, when using capillaries, it is very difficult to maintain the frozen condition--necessary for success--during the entire course of the process. Due to their low heat capacity the capillaries on the one hand tend, after the freezing, to thaw again already on their path to the deep-freezing unit while, on the other hand, the ratio of heat capacitance to energy of sublimation withdrawn from the dry material is so unfavorable that without special cooling it is impossible to prevent thawing of the content of the capillary during the drying itself. Such additional cooling measures--such as for example the use of a cooled setting surface--are however difficult to employ, due to the unfavorable shape of the capillaries and the fact that they are open at both ends, so that freeze-drying as a whole does not provide an answer.